Invasive aspergillosis (IA), caused mainly by the filamentous fungal pathogen Aspergillus fumigatus, is a severe and life-threatening infection of immunocompromised individuals. To cause invasive disease, A. fumigatus must be able to sense and utilize tissue-specific nutrient sources and effectively handle host-induced stress. A strong history of published research implicates protein kinases as essential for orchestration of a wide variety of nutrient sensing/utilization and stress response networks in pathogenic fungi. Reversible protein phosphorylation regulates almost all eukaryotic processes and, on average, about 30% of cellular proteins are modified by phosphorylation. Although no systematic analysis has yet been accomplished in A. fumigatus, the relatively few protein kinases that have been characterized play diverse roles in cellular stress responses and virulence. Furthermore, kinases are considered the second largest protein class currently functioning as drug targets, as their inhibition can be readily accomplished by small molecules. Unfortunately, the vast majority of A. fumigatus protein kinases remain unstudied. We have successfully adapted a novel CRISPR/Cas9-based mutational approach for use in wild type strains of A. fumigatus. Our preliminary data show that this facile system increases the typically low levels of gene targeting in wild type A. fumigatus to as high as 90%. With this new tool, we propose to systematically delete and functionally analyze all putative protein kinases in the wild type A. fumigatus genetic background, Af293. In Aim 1, the CRISPR/Cas9 components will be designed based on our preliminary results and purchased from commercial vendors. The necessary ribonucleotide complexes will be assembled via a short, in vitro reaction and then mixed with microhomology repair templates before protoplast transformation. Repair templates will be designed to incorporate signature tags into each kinase mutant, barcoding the strains for competitive fitness analyses. Essential kinases will be confirmed by tetracycline-regulatable promoter replacement using a modification our CRISPR/Cas9 approach. In Aim 2, we will perform competitive fitness analyses employing signature-tagged pools of kinase mutants to identify novel roles for the kinome during pathogenic growth. To identify kinases that may regulate fitness in response to host immune status, we will utilize two highly characterized models of invasive aspergillosis that re-capitulate the immune dysfunction in both neutropenic and non-neutropenic hosts. We will also perform in vitro competitive fitness assays using culture conditions that mimic pathobiologically relevant stress. A subset of the least-fit mutants from both in vivo and in vitro studies will be complemented and employed in single infection/inoculation studies to confirm roles in fitness. We expect to discover multiple, novel contributions of protein kinases to the pathobiology of invasive aspergillosis. The information generated by completion of this work will support future applications exploring novel aspects of A. fumigatus virulence.